Despite historic controversy, it has been established that Coley's toxins possessed anti-tumor effects when used in the treatment of solid tumors. "Coley's Toxins" did not in fact contain toxins, but probably contained bacterial products within the lysates that activated anti-tumor immune mechanisms. Unmethylated CpG oligodeoxynucleotides (ODN) are present in bacterial DNA and display potent immune stimulatory properties. CpG ODN could potentially link the pioneering work of Dr. William Coley's toxins to modern day cancer immunotherapy. Systemic immunity against tumor cells can be generated by stimulating antigen presenting cells (APC's) to take up exposed tumor antigens. Once primed, these APC's can differentiate and migrate from the tissues to the afferent lymphatics. In conjunction with cytokines, membrane bound ligands and other co-stimulatory molecules, the APC's activate tumor specific cytotoxic T-cells. In addition to this systemic immunity, local immunity can be induced by direct activation of natural killer cells in the tumor bed. Together, these systemic and local immune mechanisms can act synergistically in the destruction of established tumor but are often ineffective due to the tumor's ability to evade normal host immunity. We propose that efficiently presenting tumor antigens and stimulating signaling pathways that amplify the host's immune response can induce effective anti-tumor immunity. In order to amplify these signaling pathways in vivo and generate systemic immunity, we will utilize a novel vaccine strategy consisting of potent Toll-like receptor 9 (TLR-9) stimulating CpG oligodeoxynucleotide (ODN) motifs admixed with autologous irradiated tumor cells expressing GM-CSF. Synergistic cytokine immune-modulators (IL-12 and TNF :) and agents amplifying cytotoxic T-cell activation will be used to enhance and maximize the anti-tumor response. CpG ODNs have distinct immune properties and by combining various CpG motifs for tumor therapy, natural killer cells will be targeted in the tumor bed to exploit their local anti-tumor effects. The goals of this proposal are twofold. Firstly, we will define the optimal therapeutic vaccine strategy and secondly we will characterize the mechanisms by which the vaccine mediates its anti-tumor effect in non-immunogenic murine tumor models. Immunologic and molecular techniques, including functional cell assays, transgenic and knockout mouse models will be used to delineate the mechanistic action of this vaccine strategy on innate and acquired immunity. The phenotype and kinetics of immune effector cells contributing to this vaccine's immune response will be defined. Experiments will also evaluate this vaccine as an adjuvant to surgery in murine tumor models of established disease. Utilizing bacterially-derived immunogenic products to stimulate the host immune system possesses great potential for immunotherapy. To date, this form of alternative therapy has not achieved widespread clinical use and the following proposal will help define its role in tumor vaccine development. The work proposed in mice should serve as proof that this concept could potentially translate into a cellular vaccine therapy tailored to a patient's own tumor.